A neutrophil-based bone marrow-targeted drug delivery carrier and its application

Abstract

The present invention provides a neutrophil-based bone marrow-targeted drug delivery carrier and its application. The bone marrow is the apoptosis site of neutrophils, and a large number of neutrophils enter the bone marrow and undergo apoptosis every day. Free drugs or drug-loaded nanocarriers are combined with neutrophils by endocytosis by neutrophils or physical / chemical grafting on the surface. Neutrophils cultured in vitro can be used as drug carriers to achieve bone marrow-targeted delivery of drugs in vivo. The drugs can effectively penetrate vascular endothelial cells through neutrophils and enter the bone marrow, effectively increasing the drug concentration at the bone marrow site, thereby improving the therapeutic effect of bone-related diseases and achieving drug-targeted delivery at the bone marrow site.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and relates to a neutrophil-based bone marrow targeting drug delivery carrier and its application. It is achieved by obtaining drug-loaded bone marrow-targeting neutrophils and their application in the preparation of anti-tumor, antibacterial, anti-osteoporosis, anti-inflammatory and other drugs. Background Art

[0002] Bone diseases, such as bone cancer, osteomyelitis, osteoporosis and osteoarthritis, are clinically common. However, due to the characteristics of low bone blood perfusion (blood-bone marrow barrier) ([1] K. Ramanlal Chaudhari, A. Kumar, V. K. Megraj Khandelwal, M. Ukawala, A. S. Manjappa, A. K. Mishra, J. Monkkonen, R. S. Ramachandra Murthy, Bone metastasis targeting: A novel approach to reach bone using Zoledronate anchored PLGA nanoparticle as carrier system loaded with Docetaxel, Journal of Controlled Release 158(3) (2012) 470 - 478.), the treatment problems of bone diseases have not been completely solved. Therefore, it is difficult to obtain an effective drug concentration in bone after administration unless higher doses are used, which in turn leads to an increase in toxic side effects. To solve this problem, in the past few decades, many different-sized nanoparticles with different compositions have been widely explored to encapsulate therapeutic drugs to promote bone marrow targeting, but the bone marrow targeting ability of these nanoparticles is still greatly limited by the blood-bone marrow barrier and they cannot actively cross vascular endothelial cells to enter the bone marrow.

[0003] Neutrophils are the most common white blood cells in the human body, accounting for 50 - 70% of the circulating white blood cells in the body. 60% of the progenitor cells in the bone marrow differentiate into neutrophils. Generally speaking, adults produce approximately 100 billion neutrophils every day. To maintain homeostasis, the same number of senescent cells must be replaced regularly. Therefore, the half-life of neutrophils is relatively short, about 19 hours, followed by senescence and apoptosis. When neutrophils age, CXCR4 increases while CXCR2 decreases. Under the influence of the CXCR4 / CXCL12 signal, aged neutrophils migrate to the bone marrow for apoptosis ([2] C. Martin, P. C. Burdon, G. Bridger, J. C. Gutierrez-Ramos, T. J. Williams, S. M. Rankin, Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence, Immunity 19(4)(2003)583 - 93. [3] J. Wang, M. Hossain, A. Thanabalasuriar, M. Gunzer, C. Meininger, P. Kubes, Visualizing the function and fate of neutrophils in sterile injury and repair, Science 358(6359)(2017)111 - 116. [4] R. C. Furze, S. M. Rankin, Neutrophil mobilization and clearance in the bone marrow, Immunology 125(3)(2008)281 - 8.). Therefore, drug targeting delivery to the bone marrow can be achieved by means of neutrophils. Summary of the Invention

[0004] The first object of the present invention is to provide a bone marrow targeting drug delivery carrier based on neutrophils, which is specifically achieved through the following steps:

[0005] (1) Take mouse bone marrow or blood. First, use red blood cell lysate to remove red blood cells in the sample. Then, prepare a density gradient layer with percoll solution. In a centrifuge tube, carefully lay 78%, 65% and 55% percoll solutions from bottom to top respectively. Add the sample to be sorted on the top layer. After centrifuging for 20 minutes at a centrifugal force of 400g, carefully aspirate the cells between the 78% and 65% density layers. The cells in this layer are neutrophils. Verified by flow cytometry, the isolated cells are neutrophils positive for Ly6G, or double positive for Gr1 and MAIR4;

[0006] (2) Poly(lactic-co-glycolic acid) PLGA (molecular weight 10,000 - 200,000) and cabazitaxel are dissolved in dichloromethane. Then, quickly add the dichloromethane solution to a 2% PVA solution and sonicate with a probe at 30% power for 3 minutes. Next, slowly add the obtained emulsion to 0.2% PVA and stir to volatilize and remove the organic solvent. Freeze-dry to obtain drug-loaded nanoparticles;

[0007] (3) ① Prepare neutrophil drug delivery carriers by the way of neutrophils directly endocytosing nano-carriers or drugs: Neutrophils are incubated in a serum-free culture medium in an incubator for 1 hour, then 1 mg / mL free drug / drug-loaded nanoparticles are added and continue to culture for 1 hour. Then centrifuge to remove unbound free drug / drug-loaded nanoparticles, add complete medium containing serum, resuspend and continue to culture for 1 - 24 hours to make neutrophils gradually age, so as to obtain bone marrow-targeted neutrophil drug delivery carriers. ② Anchor drugs or nano-carriers on the surface of neutrophils by physical adsorption or chemical covalent modification to prepare neutrophil drug delivery carriers: Neutrophils are incubated in a serum-free culture medium in an incubator for 1 hour, then 1 mg / mL drugs / drug-loaded nanoparticles modified with active groups such as maleimide or antibodies specifically recognizing neutrophil surface antigens are added and continue to culture for 1 hour to make the drugs / nanoparticles bind to the corresponding reactive groups or antigens on the cell surface. Then centrifuge to remove unbound free drug / drug-loaded nanoparticles, add complete medium containing serum, resuspend and continue to culture for 1 - 24 hours to make neutrophils gradually age, so as to obtain bone marrow-targeted neutrophil drug delivery carriers.

[0008] For the bone marrow-targeted neutrophil drug delivery carrier of the present invention to achieve the best bone marrow targeting effect, the in vitro culture time of neutrophils is crucial. It can be enhanced by continuing to culture neutrophils in an incubator for 1 - 24 hours. The optimal culture time is 4 - 12 hours. The processing sequence can be:

[0009] (1) Neutrophils are first incubated with drugs / drug-loaded nanoparticles to obtain drug-loaded neutrophils, and then continue to culture for 4 - 12 hours to obtain bone marrow-targeted neutrophil drug delivery carriers;

[0010] (2) Or incubate neutrophils in an incubator for 4 - 12 hours first, and then add drugs / drug-loaded nanoparticles to obtain a bone marrow-targeted neutrophil drug delivery vehicle.

[0011] In the present invention, neutrophils graft / endocytose drugs. The maximum grafting degree of nanoparticles is determined by the amount of surface active molecules or receptors, and the amount of drugs loaded by neutrophils can be adjusted according to the co-incubation concentration. Among them, the involved nano-carriers include but are not limited to PLGA nanoparticles / microspheres, PS nanoparticles / microspheres, liposomes / lipid nanoparticles, emulsions, micelles, inorganic and metal nanoparticles.

[0012] The second object of the present invention is to provide the application of the said drug delivery vehicle in the preparation of drugs for treating bone-related diseases, and the bone-related diseases include bone tumors and bone marrow metastatic tumors, osteomyelitis, bone marrow bacterial infections, osteoporosis, osteoarthritis, etc.

[0013] The present invention utilizes the characteristic that neutrophils can return to the bone marrow after senescence. By encapsulating or grafting anti-tumor, antibacterial, anti-inflammatory, anti-osteoporosis and other drugs, the effective concentration of related drugs in the bone marrow is increased to treat bone-related diseases such as bone tumors and bone marrow metastatic tumors, osteomyelitis, bone marrow bacterial infections, osteoporosis, osteoarthritis, etc.

[0014] At present, there are still many problems in bone marrow-targeted drug delivery, and the main improvement method at the present stage is aimed at the design of nanoparticles. However, due to the low perfusion of bone marrow blood and the existence of the blood-bone marrow barrier, a bone marrow-targeted drug delivery vehicle with a completely new mechanism is needed. The bone marrow-targeted drug delivery vehicle based on neutrophils provided by the present invention utilizes the characteristic that senescent neutrophils highly express CXCR4 and actively return to the bone marrow, enabling drugs to hitch a ride on neutrophils, effectively penetrate vascular endothelial cells and enter the bone marrow, effectively increasing the drug concentration at the bone marrow site, thereby improving the treatment effect. Description of the Drawings

[0015] Figure 1 It is a scanning electron micrograph of cabazitaxel PLGA nanoparticles.

[0016] Figure 2 It is a neutrophil purity identification diagram.

[0017] Figure 3 It is an electron micrograph of neutrophils that have phagocytosed nanoparticles.

[0018] Figure 4 It is a fluorescence imaging diagram of neutrophils that have phagocytosed nanoparticles.

[0019] Figure 5 It is the influence of the incubation concentration of nanoparticles on neutrophil uptake.

[0020] Figure 6 is the biodistribution of neutrophils encapsulating cabazitaxel-loaded PLGA nanoparticles.

[0021] Figure 7 is the drug content in the bone marrow of neutrophils encapsulating cabazitaxel-loaded PLGA nanoparticles.

[0022] Figure 8 is the therapeutic effect of neutrophils encapsulating cabazitaxel-loaded PLGA nanoparticles on breast cancer bone metastases.

[0023] Figure 9 is the fluorescence image of the in vivo distribution of neutrophils encapsulating teriparatide-loaded PLGA nanoparticles.

[0024] Figure 10 is the therapeutic effect of neutrophils encapsulating teriparatide-loaded PLGA nanoparticles on osteoporosis.

[0025] Figure 11 is the verification of the bone marrow targeting of neutrophils encapsulating free 18F-FDG drug.

[0026] Figure 12 is the Elisa test result of the concentration of the main inflammatory factor IL-6. Detailed implementation mode

[0027] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0028] Example 1 Construction and evaluation of a bone marrow-targeted drug delivery carrier of neutrophils encapsulating cabazitaxel-loaded PLGA nanoparticles

[0029] Precisely weigh PLGA and cabazitaxel and dissolve them in dichloromethane. Then, quickly add the dichloromethane solution to a 2% PVA solution, and probe ultrasound at a power of 30% for 4 minutes with a probe. Next, slowly add the obtained emulsion to 0.2% PVA and stir to volatilize and remove the organic solvent. Freeze-dry to obtain drug-loaded nanoparticles;

[0030] Take mouse bone marrow or blood. First, use erythrocyte lysate to remove erythrocytes in the sample. Then, configure a density gradient layer with percoll solution. In a centrifuge tube, carefully lay 78%, 65% and 55% percoll solutions from bottom to top, and add the sample to be sorted on the top layer. After centrifuging at a centrifugal force of 400g for 20 minutes, carefully aspirate the cells between the 78% and 65% density layers and verify the purity of neutrophils.

[0031] Neutrophils were incubated in a serum-free culture medium in an incubator for 1 hour, then 1 mg / mL drug-loaded nanoparticles were added, and the incubation continued for 1 hour. Subsequently, the unbound free nanoparticles were removed by centrifugation. A culture medium containing serum was added, and after resuspension, the cells were further cultured in a 37°C cell incubator for 3 hours to obtain drug-loaded bone marrow-targeted neutrophils.

[0032] Morphological confirmation of paclitaxel-loaded PLGA nanoparticles (CTX-NPs): The morphology of CTX-NPs was confirmed by scanning electron microscopy ( Figure 1 ). It was shown that the surface of the nanoparticles was smooth and the particle size was uniform.

[0033] Purity confirmation of neutrophils: Flow cytometry was used to detect the purity of neutrophils in the obtained cells. The cells were stained with anti FITC-Gr1 and anti PE-MAIR4. The results showed that the concentration of neutrophils exceeded 90% ( Figure 2 ).

[0034] Confirmation of neutrophils encapsulated with paclitaxel-loaded PLGA nanoparticles (CTX-NPs@NE): Transmission electron microscopy was used to confirm the morphology of CTX-NPs@NE ( Figure 3 ). In the local magnified image, the area pointed by the arrow was the nanoparticles phagocytosed by neutrophils. Confocal microscopy was used to confirm the successful preparation of CTX-NPs@NE again. The cell nucleus, cell membrane of neutrophils and nanoparticles were labeled respectively. Under confocal microscopy, the fluorescence of the nanoparticles entered into the interior of neutrophils, indicating the successful preparation of CTX-NPs@NE ( Figure 4 ).

[0035] The concentration of nanoparticles can affect the uptake amount of neutrophils. Therefore, we labeled the nanoparticles with DiD and investigated the effect of different nanoparticle concentrations on neutrophil uptake. At the same incubation time, higher concentrations of nanoparticles could result in higher fluorescence intensity of neutrophils, indicating that neutrophils took up more nanoparticles. A nanoparticle concentration of 1 mg / mL could enable approximately 94.6% of neutrophils to take up nanoparticles ( Figure 5 ).

[0036] In vivo distribution and bone marrow targeting verification of neutrophils encapsulated with paclitaxel-loaded PLGA nanoparticles: CTX-NPs and CTX-NPs@NEs were labeled with DiR and infused into the body respectively. After 12 hours, the mice were sacrificed, and the main organs and skeletons were observed for fluorescence distribution using a small animal imaging system. The results showed that compared with free nanoparticles, the nanoparticles encapsulated by neutrophils had a reduced distribution in the liver and an increased distribution in the bone marrow ( Figure 6 ).

[0037] Verification of the increase in cabazitaxel concentration in bone marrow by neutrophils encapsulated with cabazitaxel-loaded PLGA nanoparticles: Cabazitaxel free drug (CTX), PLGA nanoparticles of cabazitaxel (CTX-NPs), and neutrophils encapsulated with cabazitaxel-loaded PLGA nanoparticles (CTX-NPs@NEs) with equal drug concentrations were respectively infused into the body through the tail vein. At 6 hours, 24 hours, and 48 hours, the mice were sacrificed respectively, the bone marrow was extracted, and the drug content was determined by high performance liquid chromatography (HPLC). The results showed that the concentration of cabazitaxel in the bone marrow of the mice in the CTX-NPs@NEs group was significantly increased compared with the other two groups ( Figure 7 ).

[0038] Anti-metastatic tumor effect in vivo: 4T1-Luc breast cancer tumor cells transfected with luciferase were inoculated into the bone marrow cavity. The Saline group, CTX free drug group, CTX-NPs group, single neutrophil NEs group, and CTX-NPs@NEs group were set up. The specific administration methods are shown in Figure 8 A. The leg circumference of the mice was measured every other day, and the tumor growth of the mice was recorded by bioluminescence imaging every week. The results showed that the tumors in the CTX-NPs group were significantly inhibited ( Figure 8 ). After the treatment was over, the mice were sacrificed, and the leg bones of the mice were observed by micro CT. The results showed that only the leg bone morphology of the mice in the CTX-NPs@NEs group remained relatively intact, and the bone density decreased less.

[0039] Example 2 Construction and evaluation of a bone marrow-targeted drug delivery carrier of neutrophils encapsulated with teriparatide-loaded PLGA nanoparticles (PTH-NPs@NEs)

[0040] Accurately weigh teriparatide and dissolve it in 50 μL of water. Then accurately weigh PLGA and dissolve it in about 500 μL of dichloromethane. Sonicate with a probe at 30% power for 3 minutes, and then add 2 - 5 mL of 2% PVA solution. Continue to sonicate with a probe at 30% power for 4 minutes. Then, slowly add the obtained emulsion to 0.2% PVA, stir and volatilize to remove the organic solvent. Lyophilize to obtain the drug-loaded nanoparticles. Obtain neutrophils according to the method of Example 1, and obtain neutrophils encapsulated with teriparatide-loaded PLGA nanoparticles (PTH-NPs@NEs).

[0041] In vivo distribution and bone marrow targeting verification of neutrophils loaded with teriparatide PLGA nanoparticles: DiR was used to label PTH-NPs and PTH-NPs@NEs, which were respectively transfused into the body. After 12 hours, the mice were sacrificed, and the main liver, spleen and bone marrow were collected and ground into single-cell suspensions, and the fluorescence signals in each organ were observed under a fluorescence inverted microscope. The results showed that compared with free nanoparticles, the nanoparticles loaded with neutrophils reduced the distribution in the liver and increased the distribution in the bone marrow and spleen( Figure 9 ).

[0042] In vivo anti-osteoporosis effect: Three-month-old C57 female mice were ovariectomized 50 days in advance, and then divided into a sham operation group (Sham), a post-operation non-treatment group (OVX), a free teriparatide drug group (PTH), a PLGA nanoparticle group of teriparatide (PTH-NPs) and a neutrophil group loaded with teriparatide PLGA nanoparticles (PTH-NPs@NEs) according to the method in Figure 10 A, and drugs were administered. After 70 days of treatment, the mice were sacrificed, and the concentrations of calcium ions, osteocalcin (OCN) and cAMP in the blood were detected. The PTH-NPs@NEs group showed the best treatment effect( Figure 10 ). Then, the leg bones of the mice were observed by microCT. The results showed that only the trabecular bone of the mice in the CTX-NPs@NEs group returned to the trabecular bone condition of normal mouse leg bones, including the normalization of bone mineral density (BMD), BV / TV and Tb.Sp indexes.

[0043] Example 3 In vivo distribution PET / CT imaging verification of a bone marrow-targeted drug delivery carrier of free 18F-FDG delivered by neutrophils

[0044] Neutrophils were cultured in glucose-free culture medium for 2-3 hours. Then, the cells were collected by centrifugation and added at a density of 10 7 / mL to 10 mCi / mL 18F-FDG and incubated in an incubator for another 1 hour. After incubation, the cells were centrifuged and free 18F-FDG was washed away with PBS. The labeled cells were transfused into the mice, and the in vivo radioactive signal distribution of the 18F-FDG-labeled neutrophils was observed 2 hours and 6 hours after transfusion using a small animal PET / CT scanner. The results showed that at 2 hours, a large number of neutrophils had carried the FDG drug into the bone marrow, and it could be seen from the results at 6 hours that the amount of the drug entering the bone marrow was still increasing. Moreover, compared with the cells cultured in vitro for 24 hours, the cells cultured in vitro for 6 hours had better targeting effects( Figure 11 ).

[0045] Example 4 Construction of a bone marrow-targeted drug delivery carrier of neutrophils loaded with dexamethasone emulsion

[0046] Accurately weigh lecithin E80, medium-chain fatty acid MCT, and vitamin E, and dissolve them in 200 μL of absolute ethanol at a mass concentration ratio of 5:5:1. Then add dexamethasone at 5% of the total lipid content by mass, and perform water bath sonication until all drugs are completely dissolved. Slowly add 2 mL of water to the ethanol phase while vigorously stirring magnetically to obtain the primary emulsion. Sonicate the primary emulsion with a probe at 40% power for 4 minutes to obtain the refined emulsion loaded with dexamethasone. Centrifuge the cells in bone marrow or blood and resuspend them with 1 mL of PBS. Then add 10 μL of anti Ly6G and incubate in an incubator at 37 °C for 20 minutes. After washing off the free antibody, neutrophils are sorted by flow cytometry. The obtained neutrophils are further cultured in a cell incubator at 37 °C for 4 hours, and then co-incubated with 1 mg / mL dexamethasone emulsion for 1 hour. Centrifuge to remove the free emulsion to obtain bone marrow-targeted neutrophils encapsulated with dexamethasone emulsion.

[0047] Establish a model of osteomyelitis by injecting LPS into the bone marrow cavity of mice. Then divide the mice into a Saline group, a free dexamethasone drug group, a dexamethasone emulsion group, and a group of neutrophils encapsulated with dexamethasone emulsion. Three days after administration, sacrifice the mice, extract the bone marrow, and detect the concentration of IL-6 using an ELISA kit. The results show that the concentration of IL-6 in the mice of the group of neutrophils encapsulated with dexamethasone emulsion decreased rapidly, indicating that the acute inflammation was alleviated ( Figure 12 ).

[0048] Example 5 Construction of a bone marrow-targeted drug delivery carrier of neutrophils grafted with gold nanoparticles

[0049] React 1 mg / mL of gold nanoparticles with a particle size of 30 - 200 nm with 1 mg / mL of biotin-PEG-SH overnight, and then centrifuge at 15,000 rpm for 30 minutes to wash away the unreacted molecules. Then add an excess of streptavidin, stir for two hours, and centrifuge at the same speed to remove the unreacted streptavidin. Incubate the extracted neutrophils with DSPE-PEG-biotin for 4 hours to label the surface of the neutrophils with biotin. Then, co-incubate the reaction product and the biotin-labeled neutrophils in a cell incubator at 37 °C for 1 hour, and centrifuge to remove the free gold nanoparticles. Infuse the cells grafted with the carrier back into the body.

[0050] Example 6 Construction of a bone marrow-targeted drug delivery carrier of neutrophils grafted with paclitaxel liposomes

[0051] Paclitaxel liposomes were prepared by the thin-film dispersion method. Specifically, lecithin E80, cholesterol Chol, and DSPE-PEG-Mal were accurately weighed and added to chloroform at a mass ratio of 10:2:1. Then, paclitaxel with a lipid mass fraction of 5% was accurately weighed and dissolved therein. At 30 °C, chloroform was removed using a rotary evaporator under vacuum. 1 mL of water was added to redisperse the lipids, and then probe sonication was performed at a power of 25% for 3 minutes to obtain uniform modified paclitaxel liposomes.

[0052] Under serum-free conditions, the paclitaxel liposomes were co-incubated with the obtained neutrophils for one hour. The culture medium was pipetted and mixed every ten minutes to help the maleimide groups modified on the liposome surface connect with the free sulfhydryl groups on the neutrophil surface. Centrifugation was performed to remove the free liposomes. The neutrophils grafted with paclitaxel liposomes were further cultured in an incubator for 4 hours to obtain bone marrow-targeted drug-loaded neutrophils.

[0053] Example 7 Construction of Bone-Targeted Neutrophils Grafted with Indomethacin

[0054] The indomethacin NHS active ester was co-incubated with the extracted neutrophils in a PBS solution at pH = 6.8 for 2 hours. Then, the free indomethacin was removed by centrifugation, and the cells were further cultured in a 37 °C cell incubator for 10 hours. Then, they were transfused back into the body. As the neutrophils aged, the drug was carried into the bone marrow.

Claims

1. A neutrophil-based bone marrow-targeted drug delivery carrier, characterized in that, It is achieved through the following steps: (1) Purified neutrophils are obtained by density gradient centrifugation, flow sorting or magnetic bead sorting of bone marrow extract or blood, which are positive for Ly6G, or double positive for Gr1 and MAIR4; (2) Cabazitaxel PLGA nanoparticles are prepared by the single emulsion method. Cabazitaxel and PLGA are dissolved in dichloromethane at the same time, and then the dichloromethane solution is quickly added to a 2% PVA solution, sonicated with a probe for 3 minutes, and transferred to a 0.2% PVA solution. After the organic solvent is volatilized, it is washed and freeze-dried to obtain cabazitaxel PLGA nanoparticles with uniform particle size; (3) Drug-loaded neutrophils are obtained by co-incubation in culture medium or PBS, and the drug-loaded neutrophils are further incubated in an incubator for 1-24 hours to obtain bone marrow targeting characteristics.

2. The drug delivery carrier according to claim 1, wherein The drug-loaded neutrophils are further incubated in an incubator for 4-12 hours to obtain bone marrow targeting characteristics.

3. Use of the drug delivery carrier according to claim 1 in the preparation of a drug for treating bone-related diseases.

4. The application according to claim 3, characterized in that, The bone-related diseases include bone tumors and bone metastases, osteomyelitis, bone bacterial infections, osteoporosis or osteoarthritis.

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